Mikihiro Yoshie

Heightened uterine mammalian target of rapamycin complex 1 (mTORC1) signaling provokes preterm birth in mice

Although preterm delivery is a major global health issue, its causes and underlying mechanism remain elusive. Using mutant mice, mimicking aspects of human preterm birth, we show here that uterine decidual senescence early in pregnancy via heightened mammalian target of rapamycin complex 1 (mTORC1) signaling is a significant contributor of preterm birth and fetal death, and that these adverse phenotypes are rescued by a low dose of rapamycin, an inhibitor of mTORC1 signaling. This role of mTORC1 signaling in determining the timing of birth in mice may help us better understand the mechanism of the timing of birth in humans and develop new and improved strategies to combat the global problem of preterm birth.

Insulin-like growth factor binding protein-7 (IGFBP7) blocks vascular endothelial cell growth factor (VEGF)-induced angiogenesis in human vascular endothelial cells

Insulin-like growth factor binding protein-7 (IGFBP7) and vascular endothelial growth factor (VEGF) are expressed in vascular endothelial cells in several tumor types. In this study, we examined the effect of IGFBP7 on VEGF-induced tube formation in cultured human umbilical vein endothelial cells (HUVECs) and its potential action in the modulation of VEGF signaling in vascular cells. IGFBP7 treatment suppressed VEGF-induced tube formation, proliferation, and the phosphorylation of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) 1/2 in HUVECs. IGFBP7 attenuated VEGF-enhanced cyclooxygenase (COX)-2 and VEGF mRNA expression, and prostaglandin E(2) secretion. Knocking down endogenous IGFBP7 enhanced COX-2 and VEGF mRNA expression. A significant increase in IGFBP7-induced caspases was not observed in the presence of VEGF. These findings indicate that IGFBP7 can modulate the stimulatory effect of VEGF on angiogenesis by interfering with VEGF expression as well as VEGF signaling and not by inducing apoptosis.

Stathmin, a Microtubule Regulatory Protein, Is Associated with Hypoxia-Inducible Factor-1α Levels in Human Endometrial and Endothelial Cells

Local hypoxia that occurs during menstruation triggers angiogenesis that is crucial for cyclical remodeling of the endometrium during the menstrual cycle. Hypoxia is thought to be important for the expression of vascular endothelial growth factor (VEGF) via its transcriptional factor, hypoxia inducible factor (HIF)-1alpha, in the endometrium. The activation of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway may modulate HIF-1alpha protein levels. Stathmin, a microtubule regulatory protein, was expressed in the stroma, glandular epithelium, and vascular endothelium in human endometrium. In this study, we examined a possible role of stathmin in hypoxia-induced HIF-1alpha and VEGF expression in primary isolated and immortalized human endometrial stromal cells, glandular epithelial cells, and human umbilical venous endothelial cells (HUVEC). Knocking down stathmin expression using small interfering RNA caused microtubule stabilization and inhibited hypoxia-induced VEGF mRNA expression via the reduction of HIF-1alpha protein levels in endometrial cells and HUVEC. Treatment of the cells with a PI3K inhibitor, wortmannin, inhibited the expression of VEGF mRNA and the accumulation of HIF-1alpha protein. Silencing of stathmin expression repressed the activation (phosphorylation) of Akt in endometrial cells and HUVEC. These results suggest that endometrial stathmin is linked to HIF-1alpha protein accumulation and VEGF expression through the PI3K/Akt signaling pathway and may be involved in regeneration of the endometrium during the menstrual cycle in human uterine cells.

Possible role of the exchange protein directly activated by cyclic AMP (Epac) in the cyclic AMP-dependent functional differentiation and syncytialization of human placental BeWo cells

BACKGROUND The mononuclear villous cytotrophoblast (CTB) differentiates and fuses to the multinucleated syncytiotrophoblast (STB), which produces hCG and progesterone. cAMP-mediated intracellular pathways are involved in the process of endocrine differentiation and fusion (syncytialization). The exchange protein directly activated by cAMP (Epac) is a mediator of cAMP signaling. We examined the differential roles of Epac and protein kinase A (PKA) signaling in the cell fusion and differentiation of trophoblast-derived BeWo cells. METHODS Epac1 and Epac2 were localized in human placental tissue (n = 9) by immunohistochemistry. The PKA-selective cAMP analog (N(6)-phenyl-cAMP, Phe) or Epac-selective cAMP analog (CPT) was tested for effects on hCG and progesterone production, and syncytialization in BeWo cells. The effect of knockdown of Epac or its downstream target molecule (Rap1) on syncytialization was evaluated. RESULTS Epac1 and Epac2 proteins were expressed in villous CTB, STB, stroma, blood vessels and extravillous CTB of the placenta. Phe increased the expression of hCG alpha/beta mRNA and secretion of hCG protein in BeWo cells (P < 0.01 versus control). CPT-stimulated production of hCG (P < 0.05), albeit to a lesser extent than Phe. Progesterone production was also enhanced by Phe or CPT (P < 0.01 and P < 0.05, respectively). CPT or a stable cAMP analog (dibutyryl-cAMP: Db) increased the number of syncytialized BeWo cells (P < 0.01), whereas Phe did not stimulate fusion. CPT- or Db-induced syncytialization was observed, even in the presence of a PKA inhibitor. Knockdown of Epac1 or Rap1 repressed the Db-, CPT- or forskolin-induced cell fusion. CONCLUSIONS The Epac signaling pathway may be associated with the cAMP-mediated functional differentiation and syncytialization of human trophoblasts.

Uterine Deletion of Trp53 Compromises Antioxidant Responses in the Mouse Decidua

Preterm birth is a global health issue impacting millions of mothers and babies. However, the etiology of preterm birth is not clearly understood. Our recent finding that premature decidual senescence with terminal differentiation is a cause of preterm birth in mice with uterine Trp53 deletion, encoding p53 protein, led us to explore other potential factors that are related to preterm birth. Using proteomics approaches, here, we show that 183 candidate proteins show significant changes in deciduae with Trp53 deletion as compared with normal deciduae. Functional categorization of these proteins unveiled new pathways that are influenced by p53. In particular, down-regulation of a cluster of antioxidant enzymes in p53-deficient deciduae suggests that increased oxidative stress could be one cause of preterm birth in mice harboring uterine deletion of Trp53.

Expression of stathmin, a microtubule regulatory protein, is associated with the migration and differentiation of cultured early trophoblasts

BACKGROUND The microtubule-destabilizing protein stathmin is expressed by the villous cytotrophoblasts and invasive extravillous trophoblasts (EVTs) in the first-trimester human placenta. Here, we evaluated the significance of stathmin expression in terms of the functions of trophoblasts. METHODS We employed two choriocarcinoma cell lines (BeWo and JEG-3), an EVT cell line (HTR-8/SVneo) and isolated first-trimester trophoblast cells. The effects of small-interfering (si) RNA-mediated stathmin knockdown on trophoblast proliferation and migration were measured by WST-1 and Transwell assays, respectively. Trophoblast differentiation was induced by dibutyryl (db)-cAMP treatment and evaluated by measuring human chorionic gonadotrophin beta (hCGbeta) and syncytin expression and cell fusion. We examined the effect of knockdown and induced stathmin expression on db-cAMP-induced differentiation. RESULTS siRNA-induced silencing of stathmin expression had a marked inhibitory effect on BeWo, JEG-3 and HTR-8/SVneo cell migration and also suppressed their proliferation, albeit to a lesser extent. db-cAMP-enhanced hCGbeta and syncytin expression and cell fusion in BeWo cells was inhibited by stathmin knockdown. However, induced expression of stathmin reversed the hCGbeta and syncytin expression and cell fusion in the Tet-On BeWo cells. Suppression of stathmin expression also inhibited the migration of and hCGbeta production by first-trimester trophoblasts. CONCLUSIONS Stathmin expression may be closely associated with early trophoblast migration and differentiation into syncytiotrophoblasts during placentation.

Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac)

INTRODUCTION Human endometrial stromal cells (ESCs) undergo differentiation during the decidualization process. Decidualization is characterized by their enhanced production of IGF binding protein-1 (IGFBP-1), prolactin (PRL), and the forkhead transcriptional factor FOXO1, and transformation into more rounded cells, during the secretory phase of the menstrual cycle and subsequent pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is crucial for this process. The present study was undertaken to examine the involvement of a mediator of cAMP signaling, exchange protein directly activated by cAMP (Epac), in decidualization of cultured ESCs. RESULTS Treatment of ESCs with the Epac-selective cAMP analog 8-CPT-2-OMe-cAMP (CPT) had no effect on IGFBP-1, PRL, and FOXO1 mRNA expression. However, CPT potentiated IGFBP-1 and PRL expression stimulated by the PKA-selective cAMP analog N(6)-Phe-cAMP (Phe) and activated Rap1, a downstream regulator of Epac signaling. Knock-down of Epac1, Epac2, or Rap1 significantly inhibited the Phe- or Phe/CPT-induced increase in IGFBP-1 and PRL expression, as well as Rap1 activation. Furthermore, CPT enhanced IGFBP-1 and PRL expression and the morphological differentiation induced by ovarian steroids, whereas Epac1, Epac2, or Rap1 knock-down suppressed these events. CONCLUSION These data provide evidence for the involvement of the Epac/Rap1 signaling pathway in cAMP-mediated decidualization of human ESCs.

Stimulation of tube formation mediated through the prostaglandin EP2 receptor in rat luteal endothelial cells

To explore the role of prostaglandin E(2) (PGE(2)) in angiogenesis in the developing corpus luteum, luteal microvascular endothelial-like cells (luteal ECs) were prepared from highly luteinizing ovaries of rats using the percoll density gradient method. The cells abundantly expressed the mRNAs of the endothelial markers CD31 (PECAM-1) and responded to the vascular endothelial growth factor (VEGF) to form in vitro tube structures on Matrigel. Cyclooxygenase (COX) inhibitors significantly suppressed tube formation in luteal ECs, whereas PGE(2) counteracted the COX inhibitor-induced blockage. PGE(2)-induced tube formation was blocked by a cyclic AMP-dependent protein kinase A (PKA) inhibitor, H89. The antagonist against the PGE receptor type 2 (EP2 receptor), AH6809, completely inhibited PGE(2)-induced tube formation and partly suppressed the VEGF-induced tube formation but did not attenuate PGE(2)-induced phosphorylation of both AKT kinase and extracellular signal-regulated kinase 1/2. VEGF significantly enhanced the expression of COX-2 mRNAs detected by real-time RT-PCR and PGE(2) secretion into the media measured by ELISA in luteal ECs. In turn, PGE(2) stimulated VEGF expression. In vitro co-culture of luteal ECs with steroidogenic luteal cells (SLCs) promoted tube formation. Pre-treatment of SLCs with VEGF further enhanced tube formation of ECs, and this effect was blocked by the COX-2 inhibitor. This stimulatory effect was inhibited by treatment with AH6809. These data indicate that PGE(2) exerts a direct stimulatory effect on tube formation mainly via the EP2 receptor/PKA signaling in luteal ECs. Our results suggest the possibility that the endogenous PGE(2) that is produced from luteinizing follicular cells as well as ECs may stimulate luteal angiogenesis.

The role of exchange protein directly activated by cyclic AMP 2-mediated calreticulin expression in the decidualization of human endometrial stromal cells.

Decidualization of human endometrial stromal cells (ESCs) accompanied by the production of prolactin (PRL) and IGF-binding protein (IGFBP) 1 and rounded-cell morphology is indispensable for the establishment and maintenance of pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is known to be crucial for decidualization. We previously reported that activation of a cAMP mediator, called Exchange protein directly activated by cAMP (EPAC) promotes cAMP analog- or ovarian steroid-induced decidualization in cultured human ESCs. In addition, small interfering RNA-mediated knock-down of the EPAC subtypes, EPAC1 or EPAC2, or knock-down of Rap1, a downstream factor of EPAC signaling, blocked functional and morphological decidualization of ESCs. However, factors downstream of EPAC2 other than Rap1 have not been determined. The present study was undertaken to identify additional downstream targets of EPAC2 associated with decidualization. Using proteomic analysis, we identified calreticulin (CRT) as a potential target of EPAC2. Knock-down of CRT expression in cultured ESCs significantly inhibited PKA-selective cAMP analog- or PKA-selective cAMP analog plus EPAC-selective cAMP analog-induced PRL and IGFBP1 expression. Furthermore, CRT knock-down suppressed the ovarian steroid-stimulated PRL and IGFBP1 expression and morphological differentiation, and silencing of EPAC2 or CRT significantly increased senescence-associated β-galactosidase activity with enhanced p21 expression and decreased p53 expression. These results suggest that EPAC2 and CRT are associated with cellular senescence in ESCs. In conclusion, we demonstrate here that EPAC2-mediated CRT expression is essential for the functional and morphological differentiation of ESCs into decidual cells. Furthermore, both EPAC2 and CRT might prevent ESCs from undergoing abnormal cellular senescence during decidualization.

Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus

The optimal decidualization of endometrial stromal cells (ESCs) following embryo implantation is one of the critical steps to establish pregnancy in rodents and humans. This step is intricately regulated by ovarian hormones. Using in vitro human ESCs model, we previously showed that activation of a cAMP mediator, exchange protein directly activated by cAMP (EPAC), promotes ovarian steroid- or cAMP analog-induced decidualization. However, expressions and functions of EPAC and RAP1 in the uterus during pregnancy have not yet been examined. In this study, we found that the expression of EPAC2 and RAP1 was markedly upregulated in the decidual cells at the implantation sites on days 7 and 9 of pregnancy in rats. Furthermore, both delayed-implantation and artificial decidualization models showed that EPAC2 and RAP1 expression was enhanced in decidual cells. Significant activation of cAMP-responsive element-binding protein (CREB), a central transcriptional factor of cAMP signaling, was observed in decidual cells. These spatiotemporal expressions of protein related EPAC pathway are overlapped by sites with activated cAMP signaling, indicating the association of EPAC signaling with decidualization. Strikingly, further studies in in vitro rat decidualization model showed that the cAMP analog and medroxyprogesterone stimulated the expression of decidual markers, while knockdown of EPAC1/2 and RAP1 attenuated the expressions of these markers. Together, these findings suggest that EPAC and RAP1 are the crucial factors for endometrial decidualization in rat pregnancy.